CAP1-Mediated Actin Cycling Via ADF/Cofilin Proteins Is Essential for Asymmetric Division in Mouse Oocytes Zhe-Long Jin, Yu-Jin Jo, Suk Namgoong* and Nam-Hyung Kim*

RESEARCH ARTICLE CAP1-mediated actin cycling via ADF/cofilin proteins is essential for asymmetric division in mouse oocytes Zhe-Long Jin, Yu-Jin Jo, Suk Namgoong* and Nam-Hyung Kim*

ABSTRACT 2013; Sun et al., 2011b) represent a family of proteins that initiate Dynamic reorganization of the actin cytoskeleton is fundamental to new actin filament formation and contribute to off-center meiotic a number of cellular events, and various actin-regulatory proteins spindle positioning. Whereas FMN2 and SPIRE are mainly involved modulate actin polymerization and depolymerization. Adenylyl in the formation of the cytoplasmic actin mesh network, the ARP2/3 cyclase-associated proteins (CAPs), highly conserved actin complex (Chaigne et al., 2015, 2013; Sun et al., 2011b), JMY monomer-binding proteins, have been known to promote actin (Sun et al., 2011a) and N-WASP (Yi et al., 2011) are responsible disassembly by enhancing the actin-severing activity of the for the cortical actin cap and subcortical actin network formation. ADF/cofilin protein family. In this study, we found that CAP1 In addition to actin nucleators, other actin-binding proteins, including regulated actin remodeling during mouse oocyte maturation. capping proteins (Jo et al., 2015), tropomodulin (Jo et al., 2016), Efficient actin disassembly during oocyte maturation is essential tropomyosins (Jang et al., 2014), and the actin depolymerizing for asymmetric division and cytokinesis. CAP1 knockdown impaired factor (ADF)/cofilin protein family, have emerged as important meiotic spindle migration and asymmetric division, and resulted in factors in cytoplasmic actin mesh network maintenance in mouse an accumulation of excessive actin filaments near the spindles. oocytes. In contrast, CAP1 overexpression reduced actin mesh levels. Modulation of the dynamic balance between filamentous actin CAP1 knockdown also rescued a decrease in cofilin family protein (F-actin) and globular actin (G-actin) is a central mechanism of actin overexpression-mediated actin levels, and simultaneous expression cytoskeleton regulation. To recycle actin monomers, the F-actin of human CAP1 (hCAP1) and cofilin synergistically decreased filament is depolymerized to form G-actin. Filament severing is cytoplasmic actin levels. Overexpression of hCAP1 decreased the mediated by ADF/cofilin family proteins, which bind to the sides of amount of phosphorylated cofilin, indicating that CAP1 facilitated filaments (Bernstein and Bamburg, 2010). In addition to ADF/cofilin actin depolymerization via interaction with ADF/cofilin during mouse proteins, several other proteins are involved in efficient actin filament oocyte maturation. Taken together, our results provide evidence for recycling. One type of protein involved in the actin-severing pathway the importance of dynamic actin recycling by CAP1 and cofilin in the is the adenylyl cyclase-associated protein (CAP, also known as SRV2 asymmetric division of mouse female gametes. in yeast) (Freeman and Field, 2000). CAP was first identified as a component of the yeast adenylyl cyclase complex that regulates This article has an associated First Person interview with the first both the actin cytoskeleton and the Ras/cAMP pathway (Fedor- author of the paper. Chaiken et al., 1990; Field et al., 1990). A recent study showed that CAPs enhanced actin monomer recharging with ATP antagonistically KEY WORDS: Actin, CAP1, ADF/cofilin, Oocyte, Asymmetric division to ADF/cofilin (Ono, 2013), and they also promoted the severing of actin filaments in cooperation with ADF/cofilin (Chaudhry et al., INTRODUCTION 2013). Yeast and mammalian CAP homologues enhanced actin The actin cytoskeleton consists of highly dynamic filament arrays, filament severing by 4–8-fold (Chaudhry et al., 2013; Jansen et al., whose assembly and rapid turnover are essential during various 2014) stages of mammalian oocyte maturation, including asymmetric Two distinct mammalian CAP isoforms are known, CAP1 spindle migration, cortical actin cap formation, polar body extrusion and CAP2 (Hubberstey and Mottillo, 2002; Yu et al., 1994, 1999). and cytokinesis (Almonacid et al., 2014; Sun and Schatten, 2006; In mammals, CAP1 is expressed ubiquitously, excluding in the Yi and Li, 2012). Various actin-binding proteins play essential roles skeletal muscle, and CAP2 is predominantly expressed in the brain, in the regulation of dynamic actin filament formation, elongation heart, skeletal muscle and testes (Yu et al., 1994). Human CAP1 has and depolymerization (Pollard and Borisy, 2003; Pollard and been found to play a key role in accelerating actin filament turnover Cooper, 2009). For example, actin nucleators such as formin-2 by effectively recycling cofilin family proteins (cofilin hereafter) (FMN2) (Dumont et al., 2007), SPIRE (Pfender et al., 2011) and and actin and by interacting with both ends of the actin filament the ARP2/3 complex (also known as ARPC) (Chaigne et al., 2015, (Moriyama and Yahara, 2002). CAP1 depletion leads to alterations in the actin cytoskeleton, cofilin and FAK phosphorylation, as well as increased cell adhesion and motility, and abnormal morphology Department of Animal Sciences, Chungbuk National University, Cheongju 361-763, in various cells (Bertling et al., 2004; Zhang et al., 2013). Mouse Korea. CAP1 forms hexameric structures that autonomously bind to F-actin,

*Authors for correspondence ([email protected]; enhance cofilin-mediated severing, and catalyze the nucleotide [email protected]) exchange of actin (Jansen et al., 2014). Considering the importance of CAPs in the actin dynamics of Z.-L.J., 0000-0002-3408-0649; Y.-J.J., 0000-0002-9067-3537; S.N., 0000-0002- 4395-5558; N.-H.K., 0000-0003-1741-9118 several cells, we investigated the role of CAP1 in the actin dynamics of murine oocyte maturation, where dynamic actin organization is

Received 5 July 2018; Accepted 23 October 2018 crucial for spindle migration and asymmetric divisions. Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs222356. doi:10.1242/jcs.222356

RESULTS meiotic maturation, we depleted CAP1 mRNA by introducing CAP1 localized in the cytoplasm and at the cortical actin cap CAP1 siRNA into oocytes. We confirmed via western blotting during mouse oocyte maturation (1.21±0.1 vs 0.52±0.4; mean±s.e.m.), immunostaining, and qPCR To investigate the functional role of CAP1 in mouse oocyte analyses (1.04±0.04 vs 0.12±0.05) that siRNA introduction maturation, we first examined CAP1 subcellular localization during caused CAP1 mRNA and protein depletion (Fig. 2A–C). CAP1 maturation. Oocytes were sampled at each developmental stage, knockdown (CAP1 KD) oocytes frequently exhibited multiple corresponding to the mature germinal vesicle stage (GV), germinal polar bodies, abnormally large polar bodies, or membrane bleb vesicle breakdown (GVBD), metaphase I (MI) and metaphase II (Fig. 2D,E). Co-injection of mScarlet–hCAP1 was shown to rescue (MII). Subcellular localization of endogenous CAP1 was monitored these defects (Fig. 2D–H), with abnormal polar body extrusion via immunofluorescence staining. CAP1 showed punctate distribution rates of: control, 6.06±3.03%; CAP1 KD, 31.72±3.51%; rescue: throughout the entire cytoplasm during all oocyte developmental 13.92±3.08% (Fig. 2G). Subsequently, we examined the effect of stages (Fig. 1A). CAP1 was also enriched at the cortical actin cap CAP1 knockdown on mouse oocyte maturation. The maturation during the MI stage (Fig. 1B; Fig. S1). To confirm CAP1 localization, rate of oocytes injected with CAP1 siRNA was lower than that of we expressed exogenous mScarlet-fused human CAP1 (mScarlet– control oocytes. The percentage of oocytes that matured to the MII hCAP1) in MI mouse oocytes. mScarlet–hCAP1 was dispersed stage was significantly decreased in the CAP1-knockdown oocytes throughout the cytoplasm and at the actin cap (Fig. 1C), supporting (Fig. 2I,J), with polar body extrusion rates of: control, 87.62±4.72%; the immunostaining localization results. CAP1 KD, 58.87±8.1%; rescue, 81.89±1.1% (Fig. 2J). Moreover, the ratio of polar body diameter to oocyte diameter was significantly Knockdown of CAP1 induced meiotic arrest and impaired higher in the CAP1 knockdown oocytes, which was also rescued asymmetric division of oocytes by mScarlet–hCAP1 injection (Fig. 2K). An abnormally large polar Previously, CAP1 depletion in somatic cells has been shown to lead body was defined as having a diameter 50% greater than that of the to F-actin accumulation and cytoskeletal defects (Zhang et al., oocyte. Collectively, these results indicated that CAP1 depletion 2013). Therefore, to investigate the function of CAP1 during mouse negatively impacted mouse oocyte asymmetric division.

Fig. 1. CAP1 localization during mouse meiotic maturation. (A) Subcellular CAP1 localization during mouse oocyte meiosis, determined through staining with anti-CAP1 antibody. For the negative control, the anti-CAP1 primary antibody was omitted. CAP1 showed punctate distribution throughout the entire cytoplasm at all developmental stages. Blue, DNA; red, actin; green, CAP1. Scale bar: 20 μm. (B) Localization of CAP1 at the cortical actin cap in MI oocytes. Blue, DNA; red, actin; gray, CAP1. Scale bar: 10 μm. (C) Localization of mScarlet–human CAP1 (mScarlet–hCAP1) at MI oocytes. CAP1 was co-localized with actin in the cortical actin cap. Blue, DNA; green, actin; red, CAP1. Scale bar: 20 μm. Journal of Cell Science

2 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs222356. doi:10.1242/jcs.222356

Fig. 2. See next page for legend.

Knockdown of CAP1 caused cytoplasmic actin mesh cytoplasmic actin mesh is crucial for spindle migration during accumulation around the spindle during oocyte maturation meiosis (Azoury et al., 2011, 2008). Therefore, we performed time- and impaired spindle migration lapse microscopy to investigate the effects of CAP1 knockdown on Increased polar body size and symmetrical division is one of spindle migration and dynamic cytoplasmic actin filament changes. the main phenotypes associated with spindle migration failure in The spindle moved from the center to near the cortex in control

MI oocytes (Namgoong and Kim, 2016). Moreover, a dynamic oocytes; however, in CAP1-knockdown oocytes, the spindle did not Journal of Cell Science

3 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs222356. doi:10.1242/jcs.222356

Fig. 2. CAP1 is essential for asymmetric division during oocyte cytoplasmic actin mesh (Fig. 4B,C). The rate of cytokinesis failure in maturation. (A) Knockdown of CAP1 mRNA using siRNA injection. CAP1 CAP1-knockdown oocytes was significantly higher than in the immunostaining in negative control siRNA-injected (control) and CAP1 siRNA- control group (control, 0±0%; CAP1 KD, 17.67±1.45%) (Fig. 4D). injected MII oocytes shows CAP1 protein depletion in CAP1-knockdown oocytes. Green, CAP1; blue, DNA; red, actin. Scale bar: 20 μm. (B) Western Collectively, these data suggest that CAP1 knockdown negatively blotting showed depletion of CAP1 protein levels in CAP1-knockdown oocytes affects cytokinesis. compared with the control. Each lane contains protein extracted from 200 oocytes. Relative intensity of CAP1 was assessed using densitometry. Overexpression of full length CAP1 decreased cytoplasmic Normalized expression of CAP1 is quantified from two independent actin mesh levels P experiments. *** <0.001. (C) CAP1 mRNA levels in siRNA-injected oocytes To provide functional evidence for the role of CAP1 in actin (n=20) expressed relative to those in the negative control. Data in B,C indicate remodeling during oocyte maturation, we overexpressed human the mean±s.e.m. for three independent experiments. (D) The incidence of – polar body extrusion (PBE) was quantified in the absence of milrinone after CAP1 as a fusion protein with mNeongreen (mNeongreen hCAP1) 13 h and is shown as representative images. Arrows indicate abnormal polar and evaluated its effect on oocyte maturation and cytoplasmic body extrusions. Scale bar: 50 μm. (E) Representative images of normal actin mesh density. The maturation rate of oocytes expressing (control and rescue) and unusual (in response to CAP1 knockdown) oocyte mNeongreen–hCAP1 was not significantly different than that of morphologies at the MII stage are shown. Scale bar: 20 µm. (F–H) CAP1 control oocytes (Fig. 5A,B). However, CAP1 overexpression either western blot band intensity (F), rates of abnormal PBE (G), and ratio of increased the number of oocytes with multiple polar bodies or caused abnormal morphologies (H) were quantified in control, CAP1-knockdown and CAP1-rescued oocytes. *P<0.05; ***P<0.001. (I,J) Timing (I) and rate (J) of oocytes to undergo asymmetric division, similar to the phenotypes PBE were determined in control, CAP1-knockdown and CAP1-rescued observed in the CAP1-knockdown oocytes, with abnormal polar body oocytes. *P<0.05; N.S., not significant. (K) The distribution of polar body extrusion rates of: control, 4.97±1.63% (mean±s.e.m.); mNeongreen– diameter (Oa) to oocyte diameter (Ob) ratio in control, CAP1-knockdown and hCAP1, 11.97±2.66% (Fig. 5C). Because CAP1 depletion can CAP1-rescued oocytes. ***P<0.001; N.S., not significant. The experiment was increase the cytoplasmic actin mesh density, even in an abnormal – performed in triplicate and data in G J are expressed as the mean±s.e.m. In F, mesh, we examined whether CAP1 overexpression can reduce the K, box represents the 25–75th percentiles, and the median is indicated, whiskers show the range. The number of oocytes analyzed is specified in amount of cytoplasmic actin mesh. MI oocytes were collected and brackets. stained with phalloidin (Fig. 5D). Oocytes overexpressing full-length CAP1 had significantly reduced cytoplasmic actin mesh levels move or moved extremely slowly, resulting in cytokinesis failure (Fig. 5E). Moreover, cytokinesis failure was observed in CAP1- or 2-cell-like oocytes (Fig. 3A). Based on the average spindle overexpressed oocytes (control, 0±0%; mNeongreen–hCAP1, 9.56 movement in control (n=17) and CAP1-knockdown (n=22) oocytes ±0.62%) (Fig. 5F,G). Using time-lapse imaging, we found in some (Fig. 3B,C), it is evident that spindle migration speeds increased oocytes overexpressing CAP1 that cytokinesis failed, but the between8hand9hincontroloocytes.Incontrast, CAP1- chromosomes segregated and reassembled (Fig. 5H; Movies 4, 5), knockdown oocytes showed substantially delayed migration or similar to phenotypes observed in some CAP1-knockdown oocytes were immobile. We confirmed the delay in spindle movements of shown in Fig. 4C. These results further support that proper CAP1 CAP1-knockdown oocytes by measuring the distances between levels are essential to maintain the cytoplasmic actin mesh and to the spindle and cortex 9 h after the resumption of oocyte maturation ensure cytokinesis during oocyte maturation. and through spindle immunostaining (Fig. 3D,E). Further, to investigate the effect of CAP1 knockdown on cytoplasmic actin Interaction of CAP1 with cofilin is required to maintain levels, control and CAP1-knockdown oocytes at the GVBD, MI and cytoplasmic actin mesh level MII stages were sampled and stained with phalloidin. We found an ADF/cofilin is a family of actin-binding proteins that cooperate with abnormally high level of actin mesh accumulation around the spindle CAP1 and facilitate actin filament turnover (Moriyama and Yahara, in CAP1-knockdown oocytes (Fig. 3F). The amount of cytoplasmic 2002). Previously, non-phosphorylated cofilin levels were shown to actin mesh in CAP1-knockdown oocytes was significantly increased be increased through inhibiting rho-associated kinase (ROCK), during the GVBD, MI and MII stages (Fig. 3G). These CAP1- which impaired oocyte maturation (Duan et al., 2014), indicating a knockdown-mediated actin accumulations were rescued by co- role of cofilin in oocyte maturation. However, the relationship injection with mScarlet–hCAP1 at the MI stage (Fig. 3H,I). These between cofilin and its interaction partner CAP1 has not been results indicate that the CAP1 knockdown impairs spindle migration investigated in mouse oocytes. and induces abnormal cytoplasmic actin mesh accumulation. Because CAPs enhance the actin-severing activity of cofilin in vitro (Jansen et al., 2014), CAP1 knockdown in oocytes may Disruption of cytokinesis by CAP1 knockdown complement excessive expression of active (S3A mutant) cofilin, during meiosis I which effectively decreases actin levels (Wang et al., 2017). To gain more insight into how CAP1 knockdown disrupts cytokinesis, We examined the amount of actin mesh under various conditions: time-lapse imaging of maturing oocytes with the presence of an CAP1 knockdown, constitutively active cofilin overexpression, actin probe (GFP–UtrCH) was performed after GVBD. In control and both CAP1 knockdown and cofilin overexpression. As expected, oocytes, chromosomes migrated close to the cortex and polar body CAP1-knockdown oocytes showed increased actin accumulation, extrusion (Fig. 4A; Movie 1). In CAP1-knockdown oocytes, whereas cofilin-overexpressing oocytes showed decreased cytoplasmic chromosomes segregated and initiated cytokinesis, resulting actin levels. Upon simultaneous CAP1 knockdown and cofilin in multiple polar bodies and two-cell-like oocytes (Fig. 4A; overexpression, actin levels increased as compared with cofilin- Movies 2, 3). Moreover, we found that in some oocytes, cytokinesis overexpressing oocytes, but not as much as in knockdown-only occurred and the cleavage furrow was not completely formed, oocytes (Fig. 6A,B). These data indicate that the CAP1 knockdown- resulting in the reincorporation of the protruding membrane into mediated increase in actin levels is related to cofilin activity. the oocyte, which resembled a single-cell-like morphology. We also tested the relationship between cofilin and CAP1 using Cytokinesis failure in CAP1-knockdown oocytes caused segregated the ROCK inhibitor Y-27632, which effectively increases non- chromosome assembly and abnormally high levels of surrounding phosphorylated cofilin levels (Duan et al., 2014). We observed a Journal of Cell Science

4 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs222356. doi:10.1242/jcs.222356

Fig. 3. See next page for legend. similar significant decrease in the actin mesh level upon ROCK Further, we investigated the synergistic effects of mNeongreen– inhibitor (50 μM) treatment (Fig. 6C,D). However, simultaneous hCAP1 and cofilin–GFP (S3A) expression on cytoplasmic actin mesh treatment of ROCK inhibitor and CAP1 knockdown did not disassembly in mouse oocytes and found that CAP1 enhanced cofilin- show significant differences between only ROCK inhibitor-treated mediated disassembly. Simultaneous expression of mNeongreen– oocytes (Fig. 6C,D). hCAP1 and cofilin–GFP (S3A) was found to elicit a greater decrease Journal of Cell Science

5 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs222356. doi:10.1242/jcs.222356

Fig. 3. CAP1 knockdown causes accumulation of cytoplasmic actin mesh have also been shown to be detrimental to spindle migrations, as around the spindle during oocyte maturation and impairs spindle seen with actin stabilization through GFP–UtrCH overexpression migration. (A) Time-lapse microscopy of maturing control and CAP1- (Holubcová et al., 2013). The accumulation of actin filaments knockdown oocytes. GV oocytes were injection with GFP–UtrCH (actin, green) and stained with SiR-tubulin (red). Spindle locations after 2 h are marked with upon CAP1 knockdown corresponds to the role of CAP1 in yellow ovals. Scale bar: 20 μm. (B,C) Tracking of spindle migration in control facilitating the depolymerization of actin filaments. oocytes (B) and CAP1-knockdown oocytes (C). The distance (μm) of the Phosphorylated cofilin was distributed at the periphery of the spindle from its starting point at each time point is plotted. n-values are as spindle in mouse oocyte, which is consistent with previous work indicated. (D) Control and CAP1-knockdown oocytes were sampled at 8.5 h (Duan et al., 2018). We also found that CAP1 overexpression can after resumption of meiosis, and the location of spindles and DNA were decrease cofilin phosphorylation, which reduces actin mesh levels. confirmed using immunostaining of α-tubulin (green) and DNA (blue), Moreover, the expression of CAP1 and constitutively active cofilin respectively. The distance between the spindle pole and plasma membrane in vivo was marked with a red line. Dotted circle shows the oocyte boundary and white synergistically decrease actin levels, indicating that the effect line indicates diameter of the oocyte parallel to the chromosome. Scale bar: of CAP1 in mouse oocytes may be related to cofilin. The CAP1 20 μm. (E) The distances between the spindle pole and plasma membrane knockdown phenotype is partially rescued by the overexpression of were quantified in control and CAP1-knockdown oocytes. **P<0.01. (F) The cofilin, suggesting that CAP1 may facilitate actin depolymerization effects of CAP1-knockdown on the actin mesh level. Phalloidin staining of the by cofilin in mouse oocytes. cytoplasmic actin mesh at the 4 h (GVBD), 8 h (MI) and 12 h (MII). Magnified An intriguing observation from this study is that CAP1 localized views of the cytoplasmic region are shown in the squares below. Scale bars: 20 μm (white), 3 μm (red). (G) Quantification of cytoplasmic actin via phalloidin in the cortex actin cap regions of mouse oocytes and was essential fluorescence intensity at the GVBD, MI, and MII stages. *P<0.05; ***P<0.001. for actin cap formation and spindle migration. The cortical actin cap, (H) Phalloidin staining of the cytoplasmic actin mesh in control, CAP1- which forms near the approaching spindle and promotes spindle knockdown and CAP1-rescued MI oocytes. Scale bar: 20 μm. (I) Quantification positioning, contains a dense actin network (Yi et al., 2013; Yi et al., of cytoplasmic actin via phalloidin fluorescence intensity in control, CAP1- 2011). Cortical actin cap formation is dependent on the ARP2/3 P P P knockdown and CAP1-rescued MI oocytes. * <0.05; ** <001; *** <0.001. complex, Rho family GTPases, and various nucleation-promoting The experiments were performed in triplicate, in graphs in E,G,I, the box represents the 25–75th percentiles, and the median is indicated, whiskers factors (Leblanc et al., 2011; Wang et al., 2013). Previously, a show the range. The number of oocytes analyzed is specified in brackets. CAP1-null mutation and mutations in the ARP2/3 complex pathway were shown to synergistically enhance morphological defects, suggesting that CAP1 is actively involved in ARP2/3-dependent in the actin mesh level than separate expression (Fig. 6E,F), suggesting actin regulation, or that CAP1 and ARP2/3 are involved in the same a synergy between CAP1 and cofilin in the maintenance of actin process (Deeks et al., 2007). CAP1 was also shown to be localized mesh levels. We further examined the potential effects on cofilin on the actin-rich lamellipodia of migration cells (Bertling et al., phosphorylation after CAP1 overexpression. Cofilin phosphorylation 2004; Freeman and Field, 2000), where ARP2/3-mediated actin at Ser 3 was significantly decreased (Fig. 6G,H). Therefore, our results polymerization and cofilin-mediated actin depolymerization is suggest that CAP1 is required to maintain the cytoplasmic actin mesh required (DesMarais et al., 2004). These results suggested that the level through its interaction with cofilin. dynamic nature of the actin cap structure is required for fast actin nucleation and depolymerization by ADF/cofilin, and that CAP1 DISCUSSION may facilitate these processes during oocyte maturation. CAPs are evolutionarily conserved actin cytoskeleton regulatory It is clear that CAP1 is involved in actin filament depolymerization proteins with many suggested biochemical roles, including in mouse oocytes, but questions remained about how CAP1 is sequestering G-actin and enhancing nucleotide exchanges (Ono, involved in this process. The most obvious interaction candidate is 2013), enhancing actin filament severing (Chaudhry et al., 2013) ADF/cofilin, which is the main driver of actin depolymerization and enhancing actin filament oligomerization (Balcer et al., 2003). in many cells. CAP1 has been shown to displace ADF/cofilin The knockdown or knockout of CAPs have been shown to impair from ADP–actin monomers, which allowed for the recycling of cell migration in mammalian cells (Bertling et al., 2004) or ADF/cofilin for new rounds of filament depolymerization and Dictyostelium (Noegel et al., 1999). It has also been reported that actin monomers to replenish the assembly-competent pool of CAPs are involved in the actin cable assembly required for the actin (Balcer et al., 2003). The N-terminal half of mouse CAP1 endocytosis of yeast (Toshima et al., 2016). However, their functional forms a hexameric structure with six symmetrical protrusions, and role in mouse oocyte maturation, where actin remodeling-mediated has been shown to sufficiently augment ADF/cofilin-mediated spindle migration is crucial for asymmetric division, is unexplored. turnover and enhance cofilin-mediated severing by 8-fold (Jansen In this study, we provide evidence that CAP1 plays a crucial role in et al., 2014; Moriyama and Yahara, 2002). This activity was actin cytoskeleton remodeling during mouse oocyte maturation, abolished with mutations at conserved surfaces on the HFD presumably in collaboration with ADF/cofilin. domain (Quintero-Monzon et al., 2009). Our data revealed similar Depletion of CAP1 in mouse oocytes disrupted spindle migration, results to previous studies. Co-expression of mNeongreen–hCAP1 cortical actin cap formation and cytokinesis. Specifically, we observed and cofilin–GFP (S3A) elicited a greater decrease in the actin the accumulation of excessive cytoplasmic actin mesh around spindles mesh level than separate expression (Fig. 6E). After CAP1 after CAP1 knockdown. The depletion of actin nucleators responsible overexpression, cofilin phosphorylation decreased significantly for the formation of new actin filaments, such as FMN2 (Leader et al., (Fig. 6G,H), suggesting that CAP1 overexpression increases 2002), SPIRE (Pfender et al., 2011) and the ARP2/3 complex (Sun cofilin activity. Actin accumulation caused by CAP1 knockdown et al., 2011b), or actin-binding proteins responsible for the could also be rescued partially by cofilin overexpression, maintenance of actin filaments, such as capping protein (Jo et al., suggesting the possibility that some of the effect of CAP1 2015), tropomodulin (Jo et al., 2016), tropomyosin-3 (Jang et al., knockdown may be caused by cofilin. Previously, CAP1 and 2014) or filamin A (Wang et al., 2017) have been shown to impair cofilin have not been successfully co-precipitated in IP assays (Zhang asymmetric division by decreasing actin networks in oocytes. In et al., 2013). Thus, further understanding of the molecular and contrast, excessive amounts of actin filaments in the cytoplasm biochemical mechanisms of the CAP1–cofilin interaction remained Journal of Cell Science

6 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs222356. doi:10.1242/jcs.222356

Fig. 4. CAP1 knockdown impairs cytokinesis during MI in mouse oocyte. (A) Chromatin tracking of maturing control and CAP1-knockdown oocytes via time-lapse microscopy. Chromatin was visualized through the injection of cRNA encoding H2B–mCherry (magenta). The upper panel corresponds to Movie 1, the middle panel corresponds to Movie 2 and the lower panel corresponds to Movie 3. Scale bar: 20 μm. (B) Control and CAP1-knockdown oocytes were sampled at 18 h, and the cytoplasmic actin mesh and DNA were visualized using immunostaining. Scale bar: 20 μm. (C) Control and CAP1-knockdown MII oocytes were sampled and the DNA was visualized using immunostaining. Magnified views of the oocyte DNA are shown in the squares to the right (yellow). Blue, DNA; DIC, differential interference contrast. Scale bar: 20 μm(white),5μm (red). (D) The rate of cytokinesis failure in oocytes after CAP1-knockdown is increased compared to that of control oocytes. ***P<0.001. The experiment was performed in triplicate and data are expressed as the mean±s.e.m. The number of oocytes analyzed is specified in brackets. elusive. In addition, mouse N-CAP1 hexamers bind autonomously to Recently, actin filament reorganization has been implicated in F-actin, independent of cofilin. Under these conditions, N-CAP1 proper chromosome segregation during meiosis I (Mogessie and binding alters actin filament structure, leading to an increase in Schuh, 2017). Considering that CAP1 knockdown resulted in the filament crossover distance and opposing the twisting effects of accumulation of actin filaments near the spindle and chromatin, cofilin. These observations raise the possibility that N-CAP1 CAP1-mediated actin depolymerization may be required for enhances severing by introducing local discontinuities in filament the proper segregation of chromosomes and prevention of topology to accelerate cofilin-mediated fragmentation (Jansen et al., segregation errors. 2014). Moreover, another protein, twinfilin, has been found to Collectively, our data showed that CAP1-mediated actin catalyze depolymerization at the ends of actin filaments in concert depolymerization activity is essential for spindle migration in with CAP (Johnston et al., 2015), indicating the possibility of a mouse oocytes. This is the first report showing that ablation of cofilin-independent mechanism of actin depolymerization by actin depolymerization machinery impairs spindle migration in CAP. The presence and potential role of twinfilin in concert with mouse oocytes by accumulation of actin filaments, demonstrating CAP in mouse oocytes would need to be investigated further. the importance of dynamic actin filament remodeling during We also showed that hCAP1 overexpression reduced oocyte maturation. cofilin phosphorylation and decreased actin accumulation. It is not clear how hCAP1 overexpression reduced the phosphorylation MATERIALS AND METHODS levels of cofilin; however it has been suggested that the Animals accumulation of cytoplasmic actin filaments may serve as a All mice used in this study were 6–8-week-old ICR female mice. Animal mechanism to protect cells by producing abnormally high care and handling were conducted in accordance with the policies cofilin activity (Zhang et al., 2013). CAP1 may be involved in regarding the care and use of animals issued by the ethics committee cofilin activity regulation via interaction with slingshot phosphatase of the Department of Animal Science, Chungbuk National University,

(Niwa et al., 2002), which dephosphorylates and activates cofilin. Korea. Journal of Cell Science

7 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs222356. doi:10.1242/jcs.222356

Fig. 5. Overexpression of full-length CAP1 affects the cytoplasmic actin mesh levels. (A) Timing of PBE was determined in control and CAP1-overexpressing (mNeongreen–hCAP1) oocytes. (B) mNeongreen–hCAP1 was injected into GV-stage oocytes and maturation was resumed. Representative DIC image of oocytes (top left) and mNeongreen–hCAP1 expression (top right). Arrows indicate abnormal MII oocytes. Phalloidin staining of cortical actin in mNeongreen–hCAP1- injected MII oocytes (bottom). Scale bars: 20 μm(white),100μm (red). (C) Rates of abnormal PBE were quantified in control and CAP1-overexpressing oocytes. ***P<0.001. (D) Phalloidin staining of the cytoplasmic actin mesh in control and CAP1-overexpressing MI oocytes. Magnified views of the cytoplasmic region are shown in the square below. Scale bars: 20 μm(white),3μm (red). (E) Quantification of cytoplasmic actin via phalloidin fluorescence intensity in control and CAP1-overexpressing oocytes. Box represents the 25–75th percentiles, and the median is indicated, whiskers show the range. ***P<0.001. (F) Control and CAP1-overexpressing oocytes were sampled at 13 h, and the cytoplasmic actin mesh and DNAwere visualized using immunostaining. Scale bar: 20 μm. (G) The rate of cytokinesis failure in CAP1-overexpressing oocytes is increased compared to that of control oocytes. ***P<0.001. (H) Chromatin tracking of maturing control and CAP1-overexpressed oocytes via time-lapse microscopy. Chromatin was visualized through the injection of cRNA encoding H2B–mCherry (magenta). The upper panel corresponds to Movie 4 and the lower panel corresponds to Movie 5. Scale bar: 20 μm. The experiment was performed in triplicate and the data in C,G are expressed as the mean±s.e.m. The number of oocytes analyzed is specified in brackets.

Oocyte collection microinjection. Only intact GV oocytes were collected for further studies. Ovaries from 6–8-week-old female ICR mice were sliced open using a The selected oocytes were placed in M16 medium under liquid paraffin oil blade to release germinal vesicle (GV) oocytes, which were placed in at 37°C in an atmosphere containing 5% CO2 for specific incubation M2 medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with periods. All reagents and media were purchased from Sigma-Aldrich

2.5 μM milrinone to prevent germinal vesicle breakdown (GVBD) during unless otherwise stated. Journal of Cell Science

8 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs222356. doi:10.1242/jcs.222356

Fig. 6. See next page for legend.

Quantitative RT-PCR SuperScript III Reverse Transcriptase (Invitrogen). The primers used for real- Total RNA was extracted from 20 oocytes at each stage using the Dynabead time PCR (RT-PCR) were 5′-AAACCT GGC CCC TTT GTG AA-3′ (forward) mRNA DIRECT kit (Invitrogen, Grand Island, NY, USA). Corresponding and 5′-TGA CCC AGT CCA CAT GCT TC-3′ (reverse). The amplification cDNA was obtained via reverse transcription of the mRNA with a cDNA program was 95°C for 3 min followed by 35 cycles at 95°C for 15 s, 60°C for synthesis kit (Takara, Kyoto, Japan), using Oligo(dT)12–18 primers and 30 s, and 72°C for 20 s, and a final extension step at 72°C for 5 min. Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs222356. doi:10.1242/jcs.222356

Fig. 6. Functional interactions between CAP1 and cofilin. (A) Phalloidin were briefly chilled at 4°C to induce depolymerization of non-kinetochore staining of the cytoplasmic actin mesh in MI oocytes injected with control microtubules just before fixation. The antibodies used were rabbit anti-CAP1 siRNA (control), CAP1 siRNA (CAP1 KD), cofilin–GFP (S3A) and CAP1 (1:1000; ab88446, Abcam), rabbit anti-phospho-cofilin (Ser3) (1:1000; siRNA+cofilin–GFP (S3A). Magnified views of the cytoplasmic region are 77G2; Cell Signaling Technology) and anti-α-tubulin (1:500; F2168, shown in the squares below. Scale bars: 20 μm (white), 3 μm (red). Sigma-Aldrich). Secondary antibodies were conjugated with Alexa Fluor (B) Quantification of cytoplasmic actin via phalloidin fluorescence intensity in 488 (1:100; SAB4600234, Sigma-Aldrich) and Alexa Fluor 594 (1:100; P P oocytes injected with siRNA or cRNA. * <0.05; *** <0.001. (C) Phalloidin SAB4600321, Sigma-Aldrich) for 1–2 h at room temperature and washed staining of the cytoplasmic actin mesh in MI oocytes treated with control siRNA three times with washing buffer. To stain the cytoplasmic actin mesh or (control), CAP1 siRNA or ROCK inhibitor Y-27632 (50 μM). Magnified views of cortical actin, the oocytes were fixed and stained with phalloidin- the cytoplasmic region are shown in the squares below. Scale bars: 20 μm tetramethylrhodamine (10 mg/ml; Sigma-Aldrich), which labels F-actin. (white), 3 μm (red). (D) Quantification of cytoplasmic actin via phalloidin fluorescence intensity in MI oocytes. ***P<0.001. (E) Phalloidin staining The oocytes were counterstained with Hoechst 33342 (Sigma Life of the cytoplasmic actin mesh in MI oocytes injected with control siRNA Science) (10 mg/ml in PBS) for 15 min, mounted on a glass slide, and (control), cofilin–GFP (S3A), mNeongreen–hCAP1 and cofilin–GFP examined using a Zeiss LSM 710 META confocal laser-scanning (S3A)+mNeongreen–hCAP1. Magnified views of the cytoplasmic region microscope. are shown in the squares below. Scale bars: 20 μm (white), 3 μm (red). Construction of recombinant plasmids and in vitro transcriptions (F) Quantification of cytoplasmic actin via phalloidin fluorescence intensity in MI oocytes. *P<0.05; ***P<0.001; N.S., not significant (P>0.05). (G) Cofilin For mNeongreen- or mScarlet-fused hCAP1 expression, a construct encoding phosphorylation levels in control and CAP1-overexpressing (mNeongreen– the human CAP1 open reading frame was obtained from Integrated DNA hCAP1) oocytes after 8 h. Scale bar: 20 μm. (H) Quantification of Technologies (Stokie, Illinois, USA) and cloned into pRN3 vectors (Lemaire in vitro phosphorylated (p)-cofilin fluorescence intensity in control and CAP1- et al., 1995), followed by transcription using an mMessage mMachine overexpressing oocytes after 8 h. ***P<0.001. The experiment was performed T3 Kit (Thermo Fisher Scientific, Waltham, MA, USA). in triplicate, in graphs in B,D,F,H, the box represents the 25–75th percentiles, For visualization of the actin filament, GFP–UtrCH was used as an and the median is indicated, whiskers show the range. The number of oocytes F-actin probe (Burkel et al., 2007), which was obtained from Addgene analyzed is specified in brackets. (26737; Cambridge, MA, USA). pRN3-H2B-mCherry plasmids were kindly provided by Dr JungSoo Oh (Sung Kyun Kwan University, Drug treatment Suwon, Korea). A 5 mM solution of Y-27632 (Sigma-Aldrich) in water was diluted in M16 For the generation of constitutively active cofilin, the serine 3 residue was medium to a concentration of 50 mM. Oocytes were then cultured in mutated to an alanine residue to prevent phosphorylation (Moriyama et al., this medium for various times. Control oocytes were cultured in fresh 1996). The corresponding mutated gene was cloned into PCS2+ vectors M16 medium. (Jang et al., 2014), followed by in vitro transcription and poly(A) tailing performed with an SP6 mMessage mMachine Kit and a Poly(A) Tailing Kit siRNA injection (Thermo Fisher Scientific). Approximately 5–10 pl of 100 μM CAP1-targeting siRNA [5′-CUGUA- UGGAGACGGUUCUU (dTdT)-3′ corresponding to 274–1698 bp of Time-lapse imaging mouse Cap1 full-length sequence (NM_001301067.2), Bioneer, Daejeon, Briefly, siRNA, cRNA, cofilin–GFP (S3A), mNeongreen- or mScarlet- Korea] was microinjected into the cytoplasm of a fully-grown GV oocyte fused hCAP1, or GFP–UtrCH mRNAs were microinjected into GV oocytes. using an Eppendorf FemtoJet and a Nikon Diaphot ECLIPSE TE300 inverted After injection, the oocytes were cultured for 20 h in M16 medium microscope equipped with a Narishige MM0-202N hydraulic 3-dimensional containing milrinone to allow for exogenous protein expression. The micromanipulator. After injection, the oocytes were cultured in M16 medium oocytes were stained with 200 nM of SiR-tubulin (Cytoskeleton, Denver, containing 2 μM milrinone for 20 h to ensure knockdown of CAP1, washed CO, USA) for 5 h to visualize the spindles. Time-lapse imaging was five times for 2 min each with fresh M16 medium, transferred to fresh performed with a confocal laser-scanning microscope (Zeiss LSM 710 M16 medium, and cultured under paraffin oil at 37°C in an atmosphere META) equipped with a Plan Apochromat 406 1.2 NA water-immersion of 5% CO2 in air. Control oocytes were microinjected with 5–10 pl of objective, and a Chamlide observation chamber and incubator system AccuTarget Control siRNA (SN-1001, Bioneer). (Live Cell Instrument, Seoul, Korea).

Western blot analysis Statistical analyses A total of 200 mouse oocytes per sample were placed in 1× SDS sample Each experiment was performed in triplicate or greater. Statistical analyses buffer and heated at 99°C for 5 min. Proteins were separated using were performed with the SPSS software package (version 11.5; SPSS). The SDS-PAGE and transferred to polyvinylidene fluoride membranes in 1× significant differences between groups were analyzed using the Student’s transfer buffer. The membranes were blocked in Tris-buffered saline t-test. The data were expressed as the mean±s.e.m., and analysis of variance containing 0.1% Tween 20 (TBS-T) and 5% nonfat milk for 1 h, followed (ANOVA) was used to analyze the data. P-values less than 0.05 were by incubation at 48°C overnight with either rabbit anti-CAP1 (1:1000; considered statistically significant. ab88446, Abcam) or mouse anti-β-tubulin antibodies (1:1000; D3U1W, Cell Signaling Technology). The membranes were washed three times with Acknowledgements TBS-T (10 min each) and incubated for 1 h with horseradish peroxidase- The authors thank all group members for their insights and critical reading of the conjugated goat anti-rabbit-IgG or anti-mouse-IgG (1:1000; sc-2004 and sc- manuscript. 2005, Santa Cruz Biotechnology). Signals were detected using Pierce ECL western blotting substrate (Thermo Fisher Scientific). Competing interests The authors declare no competing or financial interests. Immunofluorescence analysis Oocytes were fixed with 3.7% paraformaldehyde (w/v) in phosphate- Author contributions Conceptualization: Z.-L.J., S.N., N.-H.K.; Methodology: S.N.; Formal analysis: buffered saline (PBS; P4417, Sigma-Aldrich) containing 0.1% polyvinyl Z.-L.J.; Investigation: Z.-L.J., Y.-J.J.; Data curation: Z.-L.J.; Writing - original draft: alcohol (PVA), followed by permeabilization with 1% Triton X-100 (v/v) S.N.; Writing - review & editing: S.N.; Supervision: S.N., N.-H.K.; Funding for 20 min at 37°C. After a 1 h incubation in blocking buffer [PBS acquisition: N.-H.K. containing 1% bovine serum albumin (BSA)], the samples were incubated with various antibodies in a blocking buffer overnight at 4°C and washed Funding three times with washing buffer (0.5% Triton X-100 and 0.1% Tween 20 in This work was supported by grants from the Rural Development Agency, Republic of

PBS-PVA). To examine the kinetochore–microtubule attachments, oocytes Korea, Next Generation Biogreen 21 Program [grant number PJ009594]. Journal of Cell Science

10 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs222356. doi:10.1242/jcs.222356

Supplementary information Leader, B., Lim, H., Carabatsos, M. J., Harrington, A., Ecsedy, J., Pellman, D., Supplementary information available online at Maas, R. and Leder, P. (2002). Formin-2, polyploidy, hypofertility and positioning http://jcs.biologists.org/lookup/doi/10.1242/jcs.222356.supplemental of the meiotic spindle in mouse oocytes. Nat. Cell Biol. 4, 921-928. Leblanc, J., Zhang, X., McKee, D., Wang, Z.-B., Li, R., Ma, C., Sun, Q.-Y. and Liu, X. J. (2011). The small GTPase Cdc42 promotes membrane protrusion during References polar body emission via ARP2-nucleated actin polymerization. MHR: Basic Sci. Almonacid, M., Terret, M.-É. and Verlhac, M.-H. (2014). Actin-based spindle Reprod. Med. 17, 305-316. positioning: new insights from female gametes. J. Cell Sci. 127, 477-483. Lemaire, P., Garrett, N. and Gurdon, J. B. (1995). Expression cloning of Siamois, a Azoury, J., Lee, K. W., Georget, V., Rassinier, P., Leader, B. and Verlhac, M.-H. Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able (2008). Spindle positioning in mouse oocytes relies on a dynamic meshwork of to induce a complete secondary axis. Cell 81, 85-94. actin filaments. Curr. Biol. 18, 1514-1519. Mogessie, B. and Schuh, M. (2017). Actin protects mammalian eggs against Azoury, J., Lee, K. W., Georget, V., Hikal, P. and Verlhac, M.-H. (2011). Symmetry chromosome segregation errors. Science 357, eaal1647. breaking in mouse oocytes requires transient F-actin meshwork destabilization. Moriyama, K. and Yahara, I. (2002). Human CAP1 is a key factor in the recycling of Development 138, 2903-2908. cofilin and actin for rapid actin turnover. J. Cell Sci. 115, 1591-1601. Balcer, H. I., Goodman, A. L., Rodal, A. A., Smith, E., Kugler, J., Heuser, J. E. Moriyama, K., Iida, K. and Yahara, I. (1996). Phosphorylation of Ser-3 of cofilin and Goode, B. L. (2003). Coordinated regulation of actin filament turnover by a regulates its essential function on actin. Genes Cells 1, 73-86. high-molecular-weight Srv2/CAP complex, cofilin, profilin, and Aip1. Curr. Biol. Namgoong, S. and Kim, N.-H. (2016). Roles of actin binding proteins in mammalian 13, 2159-2169. oocyte maturation and beyond. Cell Cycle 15, 1830-1843. Bernstein, B. W. and Bamburg, J. R. (2010). ADF/cofilin: a functional node in cell Niwa, R., Nagata-Ohashi, K., Takeichi, M., Mizuno, K. and Uemura, T. (2002). biology. Trends Cell Biol. 20, 187-195. Control of actin reorganization by Slingshot, a family of phosphatases that Bertling, E., Hotulainen, P., Mattila, P. K., Matilainen, T., Salminen, M. and dephosphorylate ADF/cofilin. Cell 108, 233-246. Lappalainen, P. (2004). Cyclase-associated protein 1 (CAP1) promotes cofilin- Noegel, A. A., Rivero, F., Albrecht, R., Janssen, K. P., Kohler, J., Parent, C. A. induced actin dynamics in mammalian nonmuscle cells. Mol. Biol. Cell 15, and Schleicher, M. (1999). Assessing the role of the ASP56/CAP homologue of 2324-2334. Dictyostelium discoideum and the requirements for subcellular localization. J. Cell Burkel, B. M., von Dassow, G. and Bement, W. M. (2007). Versatile fluorescent Sci. 112, 3195-3203. probes for actin filaments based on the actin-binding domain of utrophin. Cell Ono, S. (2013). The role of cyclase-associated protein in regulating actin filament Motil. Cytoskeleton 64, 822-832. dynamics - more than a monomer-sequestration factor. J. Cell Sci. 126, Chaigne, A., Campillo, C., Gov, N. S., Voituriez, R., Azoury, J., Umana-Diaz,̃ C., 3249-3258. Almonacid, M., Queguiner, I., Nassoy, P., Sykes, C. et al. (2013). A soft cortex is Pfender, S., Kuznetsov, V., Pleiser, S., Kerkhoff, E. and Schuh, M. (2011). Spire- essential for asymmetric spindle positioning in mouse oocytes. Nat. Cell Biol. 15, 958. type actin nucleators cooperate with Formin-2 to drive asymmetric oocyte division. Chaigne, A., Campillo, C., Gov, N. S., Voituriez, R., Sykes, C., Verlhac, M. H. and Curr. Biol. 21, 955-960. Terret, M. E. (2015). A narrow window of cortical tension guides asymmetric Pollard, T. D. and Borisy, G. G. (2003). Cellular motility driven by assembly and spindle positioning in the mouse oocyte. Nat. Commun. 6, 6027. disassembly of actin filaments. Cell 112, 453-465. Chaudhry, F., Breitsprecher, D., Little, K., Sharov, G., Sokolova, O. and Goode, Pollard, T. D. and Cooper, J. A. (2009). Actin, a central player in cell shape and B. L. (2013). Srv2/cyclase-associated protein forms hexameric shurikens that movement. Science 326, 1208-1212. directly catalyze actin filament severing by cofilin. Mol. Biol. Cell 24, 31-41. Quintero-Monzon, O., Jonasson, E. M., Bertling, E., Talarico, L., Chaudhry, F., Deeks, M. J., Rodrigues, C., Dimmock, S., Ketelaar, T., Maciver, S. K., Malhó,R. Sihvo, M., Lappalainen, P. and Goode, B. L. (2009). Reconstitution and and Hussey, P. J. (2007). Arabidopsis CAP1 a key regulator of actin organisation Dissection of the 600-kDa Srv2/CAP Complex. Roles for oligomerization and and development. J. Cell Sci. 120, 2609-2618. cofilin-actin binding in driving actin turnover. J. Biol. Chem. 284, 10923-10934. DesMarais, V., Macaluso, F., Condeelis, J. and Bailly, M. (2004). Synergistic Sun, Q.-Y. and Schatten, H. (2006). Regulation of dynamic events by interaction between the Arp2/3 complex and cofilin drives stimulated lamellipod microfilaments during oocyte maturation and fertilization. Reproduction 131, extension. J. Cell Sci. 117, 3499-3510. 193-205. Duan, X., Liu, J., Dai, X.-X., Liu, H.-L., Cui, X.-S., Kim, N.-H., Wang, Z.-B., Wang, Sun, S.-C., Sun, Q.-Y. and Kim, N.-H. (2011a). JMY is required for asymmetric Q. and Sun, S.-C. (2014). Rho-GTPase effector ROCK phosphorylates cofilin in division and cytokinesis in mouse oocytes. MHR: Basic Sci. Reprod. Med. 17, actin-meditated cytokinesis during mouse oocyte meiosis. Biol. Reprod. 90, 37. 296-304. Duan, X., Zhang, Y., Chen, K.-L., Zhang, H.-L., Wu, L.-L., Liu, H.-L., Wang, Z.-B. Sun, S.-C., Wang, Z.-B., Xu, Y.-N., Lee, S.-E., Cui, X.-S. and Kim, N.-H. (2011b). and Sun, S.-C. (2018). The small GTPase RhoA regulates the LIMK1/2-cofilin Arp2/3 complex regulates asymmetric division and cytokinesis in mouse oocytes. pathway to modulate cytoskeletal dynamics in oocyte meiosis. J. Cell. Physiol. PLoS ONE 6, e18392. 233, 6088-6097. Toshima, J. Y., Horikomi, C., Okada, A., Hatori, M. N., Nagano, M., Masuda, A., Dumont, J., Million, K., Sunderland, K., Rassinier, P., Lim, H., Leader, B. and Yamamoto, W., Siekhaus, D. E. and Toshima, J. (2016). Srv2/CAP is required Verlhac, M.-H. (2007). Formin-2 is required for spindle migration and for the late for polarized actin cable assembly and patch internalization during clathrin- steps of cytokinesis in mouse oocytes. Dev. Biol. 301, 254-265. mediated endocytosis. J. Cell Sci. 129, 367-379. Fedor-Chaiken, M., Deschenes, R. J. and Broach, J. R. (1990). SRV2, a gene Wang, Z.-B., Jiang, Z.-Z., Zhang, Q.-H., Hu, M.-W., Huang, L., Ou, X.-H., Guo, L., required for RAS activation of adenylate cyclase in yeast. Cell 61, 329-340. Ouyang, Y.-C., Hou, Y., Brakebusch, C. et al. (2013). Specific deletion of Cdc42 Field, J., Vojtek, A., Ballester, R., Bolger, G., Colicelli, J., Ferguson, K., Gerst, does not affect meiotic spindle organization/migration and homologous J., Kataoka, T., Michaeli, T., Powers, S. et al. (1990). Cloning and chromosome segregation but disrupts polarity establishment and cytokinesis in characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl mouse oocytes. Mol. Biol. Cell 24, 3832-3841. cyclase-associated protein. Cell 61, 319-327. Wang, H. Y., Guo, J., Lin, Z. L., Namgoong, S., Oh, J. S. and Kim, N.-H. (2017). Freeman, N. L. and Field, J. (2000). Mammalian homolog of the yeast cyclase Filamin A is required for spindle migration and asymmetric division in mouse associated protein, CAP/Srv2p, regulates actin filament assembly. Cell Motil. oocytes. FASEB J. 31, 3677-3688. Cytoskeleton 45, 106-120. Yi, K. and Li, R. (2012). Actin cytoskeleton in cell polarity and asymmetric division Holubcová, Z., Howard, G. and Schuh, M. (2013). Vesicles modulate an actin during mouse oocyte maturation. Cytoskeleton 69, 727-737. network for asymmetric spindle positioning. Nat. Cell Biol. 15, 937-947. Yi, K., Unruh, J. R., Deng, M., Slaughter, B. D., Rubinstein, B. and Li, R. (2011). Hubberstey, A. V. and Mottillo, E. P. (2002). Cyclase-associated proteins: CAPacity Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3- for linking signal transduction and actin polymerization. FASEB J. 16, 487-499. complex-driven cytoplasmic streaming in mouse oocytes. Nat. Cell Biol. 13, Jang, W.-I., Jo, Y.-J., Kim, H.-C., Jia, J.-L., Namgoong, S. and Kim, N.-H. (2014). 1252-1258. Non-muscle tropomyosin (Tpm3) is crucial for asymmetric cell division and Yi, K., Rubinstein, B., Unruh, J. R., Guo, F., Slaughter, B. D. and Li, R. (2013). maintenance of cortical integrity in mouse oocytes. Cell Cycle 13, 2359-2369. Sequential actin-based pushing forces drive meiosis I chromosome migration and Jansen, S., Collins, A., Golden, L., Sokolova, O. and Goode, B. L. (2014). symmetry breaking in oocytes. J. Cell Biol. 200, 567-576. Structure and mechanism of mouse cyclase-associated protein (CAP1) in Yu, G., Swiston, J. and Young, D. (1994). Comparison of human CAP and CAP2, regulating actin dynamics. J. Biol. Chem. 289, 30732-30742. homologs of the yeast adenylyl cyclase-associated proteins. J. Cell Sci. 107, Jo, Y.-J., Jang, W.-I., Namgoong, S. and Kim, N.-H. (2015). Actin-capping proteins 1671-1678. play essential roles in the asymmetric division of maturing mouse oocytes. J. Cell Yu, J., Wang, C., Palmieri, S. J., Haarer, B. K. and Field, J. (1999). A cytoskeletal Sci. 128, 160-170. localizing domain in the cyclase-associated protein, CAP/Srv2p, regulates access Jo, Y.-J., Jang, W.-I., Kim, N.-H. and Namgoong, S. (2016). Tropomodulin-3 is to a distant SH3-binding site. J. Biol. Chem. 274, 19985-19991. essential in asymmetric division during mouse oocyte maturation. Sci. Rep. 6, 29204. Zhang, H., Ghai, P., Wu, H., Wang, C., Field, J. and Zhou, G.-L. (2013). Johnston, A. B., Collins, A. and Goode, B. L. (2015). High-speed Mammalian adenylyl cyclase-associated protein 1 (CAP1) regulates cofilin depolymerization at actin filament ends jointly catalysed by Twinfilin and function, the actin cytoskeleton, and cell adhesion. J. Biol. Chem. 288, Srv2/CAP. Nat. Cell Biol. 17, 1504-1511. 20966-20977. Journal of Cell Science